Identifiying ncRNAs expressed under genotoxic stress and dormancy in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is the intracellular pathogen that causes tuberculosis (TB), an infectious disease responsible for 1.5 million deaths worldwide in 2015. This bacterium is inhaled in aerosol droplets and once in the lung, it is engulfed by alveolar macrophages. Inside the activated macrophage, Mtb is exposed to a hostile environment, including reactive oxygen species (ROS) and reactive nitrogen species (RNS) that cause oxidation, deamination and alkylation of macromolecules. This condition is fatal to most bacterial pathogens, but Mtb has the ability to sense the host environment and eventually enter into a non-replicating, persistent state. The tubercle bacilli can remain in this latent stage for a lifetime without causing disease or, under certain conditions, it can awake resulting in TB disease. In this context, the aim of our study is to identify and characterize non-coding RNAs (ncRNAs) transcribed under genotoxic stress as well as during reactivation from the dormant stage. Here, Mtb strain H37Rv was exposed to various forms of genotoxic stress (oxidative, nitrosative or alkylative stress or double strand DNA breaks) and defined culture supplements. Total RNA from treated and untreated H37Rv cells was isolated and subjected to high-throughput RNA-seq. ncRNAs were identified using Rockhopper. A subset of the ncRNAs detected was selected for a more detailed analysis, including validation by northern blot. The secondary structure of the ncRNAs under study was predicted using mfold and their potential gene targets were predicted using intaRNA and CopraRNA. Ahead, we will combine our molecular and bioinformatics results with in vitro studies, including mapping 5’ and 3’ by RACE, establishing their secondary structure with SHAPE, and detecting sRNA-mRNA interactions by performing EMSA. The potential physiological role of the Mtb ncRNAs predicted will be tested by employing mutants or by repression, deletion or over-expression of the ncRNA of interest and measuring the potential phenotypic outcome by next-generation quantitative mass spectrometry.